Phosphatidylinositol-specific phospholipase C enzymes catalyze the cleavage of phosphoinositides to diacylglycerol (a second messenger activator of protein kinase C), and inositol phosphates (cyclic 1,2-inositol phosphate in the case of bacterial PI-PLC and both cIP and inositol-1-phosphate from PI for mammalian Pl-PLC isozymes). The mammalian enzymes, key regulators of PI-signaling, have critical roles in cell growth and proliferation. The secreted bacterial PI-PLC enzymes, considerably smaller and single domain proteins, aid in infectivity of host cells. Crystal structures of two bacterial (from Bacillus thuringiensis and Listeria monocytogenes), and the smallest (85 kDa) of the mammalian isozymes, PLCdelta1 identify exposed hydrophobic residues at the rim of the (beta/alpha)8-barrel (the catalytic domain of the mammalian enzyme) that are likely to interact with membranes. We seek to characterize the mechanisms by which these interfacial residues activate and/or inhibit the enzyme activity. A combination of mutagenesis and diverse biophysical (e.g., fluorescence, magnetic resonance) methods will be used for these studies. A very novel technique, high resolution 31P field cycling, will be used to characterize the motion and conformation of substrates under conditions that kinetically activate the enzymes. The results from this work should provide a detailed picture of how the (beta/alpha)-barrel of PI-PLC enzymes interacts with the membrane surface, including how the protein alters dynamic characteristics of the substrate phospholipid, and how binding at this interfacial site is translated to enhanced catalysis. Given the importance of the mammalian PI-PLCs in signal transduction, the results may suggest new modes of modulating these activities. In the case of the PI-PLC from L. monocytogenes, an intracellular pathogen of humans that can cause serious infections in immunocompromised individuals, the work should also provide insight into how the enzyme aids in escape of the bacterium from the phagosome.